Subjects: Biology >> Bioengineering submitted time 2018-12-16 Cooperative journals: 《中国生物工程杂志》
Abstract：为了实现糖苷类物质的高效转化，将来源于副干酪乳杆菌（Lactobacillus paracasei）TK1501β-葡萄糖苷酶基因连接于表达载体pET28a(+)上，在E. coli BL21中表达，重组酶经镍离子亲和层析分离得到纯酶，其相对分子量和比酶活分别为86.63 kDa和675.56 U/mg。最适作用温度和pH分别为30 ℃和6.5。 Mg2+和Ca2+对β-葡萄糖苷酶酶活抑制作用最小，Cu2+几乎使其丧失催化活性。其底物特异性较宽泛，对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时，该酶的Km和Vmax分别为1.44 mM和58.32 mM s-1，催化系数kcat为3982 s-1。结果与分析表明来源于副干酪乳杆菌TK1501β-葡萄糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。
Subjects: Biology >> Bioengineering submitted time 2018-03-22 Cooperative journals: 《中国生物工程杂志》
Abstract：胆固醇氧化酶专一性地催化胆固醇为胆甾-4-烯-3-酮，广泛地应用于临床以及食品加工行业。本论文将来源于Pimelobacter simplex的胆固醇氧化酶PsCO4，分别转化到大肠杆菌宿主BL21(DE3)、Rosetta(DE3)和C41(DE3)中，在不同温度（15 ℃、25 ℃、37 ℃）及IPTG诱导浓度（0.01 mM、 0.1 mM、0.5 mM）下异源表达PsCO4。结果表明，转入Rosetta(DE3)菌株的PsCO4蛋白，在IPTG浓度为0.1 mM、15 ℃下经18 h诱导表达，PsCO4可溶性表达量最高(0.63 mg/ml)。异源表达的胆固醇氧化酶PsCO4最适温度为30 ℃，最适pH为7.5。通过TLC, GC-MS检测出PsCO4催化胆固醇生成胆甾-4-烯-3-酮。以胆固醇和β-谷甾醇、豆甾醇和孕烯醇酮为底物，测定PsCO4对四种底物的催化反应动力学参数，胆固醇kcat /Km 为0.08 s-1·M-1分别高于β-谷甾醇(0.04 s-1·M-1)、豆甾醇(0.005s-1·M-1)和孕烯醇酮(0.02 s-1·M-1)。
Subjects: Biology >> Bioengineering submitted time 2017-06-01 Cooperative journals: 《中国生物工程杂志》
Abstract： Cholesterol oxidase is a key enzyme during cholesterol metabolism. It could be potential used to detect the cholesterol level in serum in clinical trials. The cholesterol oxidase from Rhodococcus ruber (ChOG) was transformed into BL21(DE3) and Rosetta(DE3) for protein expression, and was induced at different conditions. The results showed that ChOG was overexpressed at 16 ℃ and 0.1 mmol/L IPTG. The activity reached 8.033 U/mg after purification with Ni-NTA superflow. The product cholest-5-en-3-one was characterized by TLC and HPLC, respectively. The structural analysis showed that Glu406, together with Arg408 and Glu261 played an important role during the dehydrogenation of cholesterol C3-OH, proton transfer, and isomerization.
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